Cdc13 Delivers Separate Complexes to the Telomere for End Protection and Replication  Erin Pennock, Kathleen Buckley, Victoria Lundblad  Cell  Volume 104, Issue 3, Pages 387-396 (February 2001) DOI: 10.1016/S0092-8674(01)00226-4

Figure 1 The DBDCDC13-STN1 Fusion Rescues the Essential Function of CDC13 (A) The DBDCDC13-STN1 fusion rescues the lethality of a cdc13-Δ null strain. Growth of cdc13-Δ/CDC13 (pVL1086), cdc13-Δ/ADH-STN1 (pVL1131), cdc13-Δ/DBDCDC13 -STN1 (pVL1253), cdc13-Δ/EST3-DBDCDC13 (pVL1292) and cdc13-Δ/vector control (pRS425) strains. Plasmids were introduced into a haploid cdc13-Δ/pVL438 (CEN URA3 CDC13) strain; growth of the viable strains corresponds to ∼25 generations after eviction of pVL438. (B) A cdc13-Δ/DBDCDC13-STN1 strain exhibits progressive telomere shortening. A genomic Southern blot, probed with poly d(GT/CA), shows the telomere length of a CDC13 strain (lane 1) or a cdc13-Δ/DBDCDC13-STN1 strain (lanes 2–4) propagated by serial culturing for ∼30, 45, or 60 generations of growth, respectively, after loss of a covering CDC13 plasmid. (C) A cdc13-Δ/DBDCDC13-STN1 strain displays a senescence phenotype. The cdc13-Δ/DBDCDC13-STN1 strain was propagated by serial culturing, and an aliquot from each successive culture was streaked onto media selecting for the plasmid and incubated at 30°C for 48 hr. The strain used in this experiment was generated independently from that shown in (B). (D) Prolonged propagation of the cdc13-Δ/DBDCDC13-STN1 strain produces type I survivors. A genomic Southern blot of a cdc13-Δ/DBDCDC13-STN1 strain, propagated by serially culturing, results in telomeric and subtelomeric rearrangements, indicative of a telomerase-independent mode of telomere maintenance. A darker exposure (left side) illustrates telomere shortening during early propagation (lanes 1 and 2, 30 and 45 generations of growth), and a lighter exposure (right side; lanes 1–5, 30 to 95 generations of growth) is shown to illustrate the amplification of sub-telomeric Y′ elements (indicated by the two arrows), which is a characteristic feature of one type of telomerase-defective survivors (Lundblad and Blackburn, 1993). In other experiments, rearrangements characteristic of the other class of survivors were observed (data not shown) Cell 2001 104, 387-396DOI: (10.1016/S0092-8674(01)00226-4)

Figure 2 The Telomere Replication Defect of a cdc13-Δ/DBDCDC13-STN1 Strain Is Restored by the Introduction of a DBDCDC13-Telomerase Fusion Protein A cdc13-Δ/pVL438 (CEN URA3 CDC13) strain was cotransformed with plasmids expressing the DBDCDC13-STN1 fusion (pVL1372) and the EST1-DBDCDC13 fusion (pVL1120), followed by eviction of the covering CDC13 plasmid and serial propagation in liquid culture. Lane 1, a CDC13 strain; lanes 2–5, a cdc13-Δ/DBDCDC13-STN1+ EST1-DBDCDC13 strain propagated for ∼30, 45, 60, 80, and 95 generations of growth following loss of the covering plasmid. The schematic figure presents an interpretation of the results in Figures 1 and 2: in the absence of intact Cdc13 protein, the minimal DBDCDC13 can be used to deliver an Stn1-containing complex (hypothetically drawn as a two-subunit complex) and the telomerase holoenzyme to the chromosome terminus to restore telomere function. Whether multiple DBDCDC13 -containing complexes can simultaneously bind to the same telomere is considered in the Discussion Cell 2001 104, 387-396DOI: (10.1016/S0092-8674(01)00226-4)

Figure 3 Reconstituting a Wild-type Telomere in the Absence of Intact Cdc13 Protein (A) Experimental plan for restoring the normal pathway for telomerase recruitment in a cdc13-Δ strain kept alive by the DBDCDC13-Stn1 fusion protein. In this strategy, telomerase access to the telomere is proposed to occur as the result of an interaction between the holoenzyme and a minimal telomerase recruitment domain of Cdc13, which is present at the telomere as a DBDCDC13-RD fusion protein. The construction of the DBDCDC13-RD fusion is also shown: the proposed recruitment domain of Cdc13p is indicated by the striped box, with the residue altered in the recruitment-defective cdc13-2 mutation (E252K) denoted by the black arrowhead, and the DNA binding domain of Cdc13p is represented by the shaded box. Note that the order of the RD and DBDCDC13 domains in the DBDCDC13-RD fusion is the opposite of that in the native protein. (B) A DBDCDC13-RD fusion protein complements the senescence phenotype of the cdc13-2 mutant. Growth after ∼75 generations of a cdc13-2 strain harboring plasmids expressing the wild-type Cdc13 protein (pVL1086), a vector control (pRS425), the DBDCDC13-RDaa190–340 fusion (pVL1269) or the DBDCDC13-RDestaa190–340 version (pVL1368). (C) A DBDCDC13-RD fusion prevents progressive telomere shortening in the cdc13-Δ/DBDCDC13-STN1 strain. A cdc13-Δ/pVL438 (CEN URA3 CDC13) strain was cotransformed with a plasmid expressing the DBDCDC13-STN1 fusion (pVL1253) and the DBDCDC13-RD fusion (present in single copy; pVL1383) and genomic DNA was prepared from serially propagated cultures. Lane 2 corresponds to ∼30 generations of growth following loss of the covering CDC13 plasmid, and each subsequent lane represents an additional ∼12 generations of growth; lane 1, a CDC13 strain. (D) A DBDCDC13-RD fusion prevents the senescence of a cdc13-Δ/pDBDCDC13-STN1 strain. A cdc13-Δ/pDBDCDC13-STN1 + pDBDCDC13-RD (2 μ; pVL1384) strain was generated and propagated by serial culturing as described in Figure 1C; the approximate number of generations following loss of the covering CDC13 plasmid is indicated Cell 2001 104, 387-396DOI: (10.1016/S0092-8674(01)00226-4)

Figure 4 The Recruitment Domain of Cdc13 Interacts with a Specific Site on the Telomerase-Associated Est1 Protein (A) The est1-60 and cdc13-2 mutations exhibit mutual suppression. A haploid est1-Δ cdc13-Δ/pVL438 (CEN URA3 CDC13) strain was cotransformed with plasmids expressing CDC13 (pVL1084) and EST1 (pVL198); CDC13 (pVL1084) and est1-62 (pVL1720); cdc13-9 (pVL1726) and est1-62 (pVL1720); cdc13-2 (pVL690) and est1-60 (pVL 1689); and CDC13 (pVL1084) and est1-60 (pVL1689). Transformants were subsequently propagated on plates containing 5-FOA to evict the covering CDC13 plasmid. The est1-60 CDC13 and est1-62 CDC13 strains are shown at their maximum senescence point (80 to 100 generations after the loss of EST1 function); the est1-60 cdc13-2, and est1-62 cdc13-9 strains are shown after propagation for an additional 50 generations. (B) Telomere length of est1 and cdc13 mutant strains. Lane 1, est1-Δ/pVL232 (EST1); lane 2, est1-Δ/YCplac33; lane 3, est1-Δ/pVL1372 (est1-60); genomic DNA was prepared from very senescent est1-Δ and est1-60 strains, which exhibit telomeric and subtelomeric rearrangements that are characteristic of late senescing est− mutants (Lundblad and Blackburn, 1993). Lanes 4–7 show the telomere length of the reciprocally suppressed cdc13-2 est1-60 double mutant strain: lane 4, CDC13 EST1; lane 5, CDC13 est1-60; and lanes 6 and 7, cdc13-2 est1-60. Lane 8, cdc13-2 EST1; lanes 9 and 11, CDC13 EST1; and lane 10, cdc13-9 EST1. (C) The Est1-60 protein retains association with telomerase. Extracts were prepared from an EST1 strain expressing Est1-ProA (containing a C-terminal protein A tag; pVL1375), Est1-60-ProA (pVL1569) or untagged Est1 (pVL232); the Est1 protein was immunoprecipitated and Northern blotting was performed to detect the telomerase TLC1 RNA subunit. Crude extract lanes (E) represent 3% of the input into the immunoprecipitation fraction (P). The efficiency of immunoprecipitation, as quantitated by PhosphorImager analysis, was 23% and 16% for Est1-ProA and Est1-60-ProA, respectively; recovery with the untagged protein was <1%. (D) The est1-60 mutation suppresses the recruitment defect of the DBDCDC13-RDest fusion protein. A cdc13-Δ/DBDCDC13-STN1 + pDBDCDC13-RDest (2 μ; pVL1386) strain that had been grown for ∼30 generations in the absence of CDC13 was transformed with either a plasmid containing the est1-60 mutation (pVL1372; lane 4) or a vector control (pRS416; lanes 2–3); lane 1, a CDC13 strain. DNA was prepared from cultures grown for ∼30 generations following introduction of the relevant plasmid Cell 2001 104, 387-396DOI: (10.1016/S0092-8674(01)00226-4)

Figure 5 Model for the Role of Cdc13 in Telomere Replication and End Protection The single-stranded G strand observed late in S phase, generated by a telomerase-independent mechanism (Wellinger et al., 1996), provides a substrate for binding by Cdc13. The processing activity that removes C strand sequences has not been identified and is shown hypothetically as a 5′ to 3′ exonuclease. We propose that binding of Cdc13 to the telomere is necessary for subsequent recruitment of two complexes that protect and replicate the telomere. The Stn1-containing end protection complex would inhibit further removal of C strand sequences, thereby protecting the telomere, followed by recruitment of telomerase by Cdc13, which elongates the G strand of the telomere. C-strand fill-in synthesis completes the process; whether Cdc13 remains bound to the telomere at this stage is not known and therefore not indicated. The negative regulatory function of Cdc13 and Stn1, which is not depicted in this figure, apparently requires a direct interaction between the two proteins (Grandin et al., 1997; Chandra et al., 2001); whether Cdc13 and Stn1 similarly execute their essential function through a direct interaction, or through an intervening additional protein (as hypothetically depicted here), is unknown Cell 2001 104, 387-396DOI: (10.1016/S0092-8674(01)00226-4)